PROJECT SUMMARY Glycans have several distinct properties that make them excellent targets for disease biomarkers. Firstly, their location glycans on cell surfaces makes them the first point of contact for cellular interactions, and thus they are crucial in the control of normal metabolic processes. Secondly, cell surface molecules are also strategically exposed for surveillance by the immune system allowing for the potential of immune recognition of abnormal cells. Thirdly, specific glycan structures that are not present, or are in low amounts, in normal states proliferate in disease states, such as cancer. And lastly, changes in glycosylation involve many proteins, including those that are highly abundant. Therefore, a single change in a cell?s glycosylation machinery can affect many different glycoconjugates. To effectively employ and discover glycan disease markers a wide range of highly-specific reagents are urgently needed. The monosaccharide fucose has been identified in many disease markers including pancreatic and prostate cancer, but is difficult to detect specifically with existing reagents. Using structurally-guided directed evolution, we will convert an ?-L-fucosidase enzyme into high affinity reagents for the detection of fucose-containing antigens. Such engineered lectin-like reagents derived from enzymes are called ?Lectenz?, and have several advantages over lectins and antibodies. The advantages of Lectenz include precise definition of specificity, tunable binding properties, and ease of recombinant expression, enabling their potential use in affinity purification, western blotting, in situ histological staining, and in vivo imaging. We will generate fucose-binding Lectenz that can differentiate between various antigenic fucose structures, such as the Lewis antigens versus core fucosylation in found in mammalian glycosylation. Glycosylation detection is essential in fully characterizing and exploiting glycans as markers of specific disease states, and yet current reagents have poor abilities to discriminate between only Lewis X or Lewis A antigens, or other Lewis antigens or core fucose. The principle advantages of an engineered Lectenz over an antibody are that the Lectenz is specific to the carbohydrate sequence, but, in contrast to antibodies, will recognize that sequence in a broad range of glycans. Further, in contrast to carbohydrate reagents based on plant lectins, engineered Lectenz are derived from enzymes that have exquisite substrate specificities and low toxicities.